Identification of long non‐coding RNA MVIH as a prognostic marker and therapeutic target in acute myeloid leukemia

Abstract Background This study aimed to investigate the correlation of long non‐coding RNA microvascular invasion in hepatocellular carcinoma (lncRNA MVIH) with disease risk, disease conditions, and prognosis of acute myeloid leukemia (AML), and also to investigate the influence of lncRNA MVIH on AML cell activities in vitro. Methods A total of 212 AML patients and 70 controls were consecutively recruited. Their bone marrow mononuclear cells (BMMCs) were isolated, and lncRNA MVIH was detected by reverse transcription quantitative‐polymerase chain reaction. In AML patients, complete remission (CR), event‐free survival (EFS), and overall survival (OS) were assessed. In vitro, lncRNA MVIH expression in AML cell lines was determined, and the effect of lncRNA MVIH on AML cell proliferation and apoptosis was assessed. Results LncRNA MVIH expression was increased in AML patients compared to controls, and receiver operating characteristic curve showed that lncRNA MVIH predicted elevated AML risk (area under curve: 0.742 [95% CI: 0.674‐0.810]). In AML patients, no correlation of lncRNA MVIH expression with French‐American‐British classification was observed, while lncRNA MVIH high expression correlated with worse risk stratification. Moreover, lncRNA MVIH expression negatively correlated with CR achievement, EFS and OS. In vitro, lncRNA MVIH was overexpressed in AML cell lines (KG‐1, ME‐1, and HT‐93) compared to normal BMMCs. Furthermore, lncRNA MVIH downregulation reduced KG‐1 cell proliferation but increased apoptosis, whereas lncRNA MVIH upregulation raised HL‐60 cell proliferation but decreased apoptosis. Conclusion LncRNA MVIH may not only serve as a prognostic marker but also act as a therapeutic target in AML.


| INTRODUC TI ON
Acute myeloid leukemia (AML), one of the most common leukemias, is characterized by abnormal proliferation of undifferentiated and non-functional leukemic blasts in the bone marrow (BM), which results in around 20% of all hematologic malignancy-related deaths. 1,2 The current progresses in AML mainly include improvements on treating opportunistic infectious diseases, advances on supportive care, and reduce of complications after allogeneic BM transplantation; however, the therapeutic effects are unsatisfactory, with a 5-year survival rate of 40% for younger patients (18-60 years) and a 5-year survival rate of 10% for the elder patients (age above 60 years), and AML is still a life-threatening disease. 1,3,4 Thus, rational development of novel strategies to improve the treatment outcomes of AML is important. Since emerging studies have revealed that the association of molecular aberrations with the prognosis may contribute to guide therapeutic decisions, exploring novel biomarkers that being identified for AML prognosis is desperately needed. 5,6 Long non-coding RNAs (lncRNAs) are key members of the noncoding RNA family that possess limited protein-coding capacity. 7 In the post-genomic era, lncRNAs have been rapidly gaining recognition for their crucial roles across diverse biological processes. 8,9 Among them, lncRNA microvascular invasion in hepatocellular carcinoma (MVIH), located on RPS24 gene and encoding a member of the S24E ribosomal proteins, has been reported to correlate with deteriorative disease progression and predict poor survival profiles in several solid tumors, such as hepatocellular carcinoma (HCC), breast cancer, and non-small cell lung cancer (NSCLC). [10][11][12][13][14] Moreover, some experiments have disclosed the ability of lncRNA MVIH to promote cell proliferation and repress apoptosis in several tumors. 10,11,14 All these data emphasized the oncogenic role of lncRNA MVIH in specific solid tumors; however, no evidence about the role of lncRNA MVIH in any hematological malignancies was found, including AML.
Considering AML had some common biological characters with solid tumors, for instance, the abnormally exuberant proliferation of malignant cells, we inferred that lncRNA MVIH, a lncRNA promoted cell proliferation in some solid tumors, might also facilitate malignant cell proliferation and contribute to disease initiation in AML.
Therefore, to validate this speculation, this study investigated the predictive value of lncRNA MVIH for disease risk and the correlation of lncRNA MVIH expression with some crucial disease conditions as well as prognosis in AML; moreover, we also explored the influence of lncRNA MVIH on AML cell activities in vitro.

| Subjects
This study consecutively recruited 212 de novo AML patients between January 2015 and December 2018. Patients were eligible for recruitment if they had (a) a diagnosis of de novo AML confirmed by examinations of morphology, immunophenotyping, cytogenetics, molecular cytogenetics, and genetics, (b) age ≥ 18 years; (c) no history of other malignancies, (iv) able to be followed up regularly.
While patients were excluded from the study if they were (a) diagnosed as acute promyelocytic leukemia (APL), (b) secondary or relapsed AML, (c) previously treated by radiotherapy, chemotherapy or stem cell transplantation before recruitment, (d) pregnant or breastfeeding women. Furthermore, the current study also enrolled a total of 70 controls including BM donors as well as subjects who underwent BM biopsy for non-hematological malignant disease.

| Ethics statement
This study was approved by Institutional Review Board of Jinhua People's Hospital and carried out in accordance with the Declaration of Helsinki. All enrolled subjects provided the written informed consents before entry to the study.

| Sample collection
Bone marrow samples of AML patients were collected before they received treatment. The BM samples of controls were collected when their eligibility was confirmed. After sample collection, the BM mononuclear cells (BMMCs) were isolated by the density gradient centrifugation. Subsequently, the lncRNA MVIH expression in the BMMCs was determined by the reverse transcription quantitativepolymerase chain reaction (RT-qPCR).

| Induction therapy and response assessment
The induction therapy decisions for AML were based on age, history of prior myelodysplasia or cytotoxic therapy, and performance status. Standard induction regimens were administered to all AML patients, and the regimen generally consisted of 3 days of an anthracycline (eg, daunorubicin 45-60 mg/m 2 or an alternative anthracycline at equivalent dose), and 7 days of cytarabine (100-200 mg/ m 2 continuous iv). Dose reduction was considered for individual patients and decided by their treating physician according to the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology of AML (version 1.2014). In addition, response assessment was commonly performed between day 21 and 28 after start of induction therapy. Complete remission (CR) was defined as BM with at least 20% cellularity and BM blasts below 5% at steady state after treatment, without cytological evidence of leukemia, no transfusion requirement, leukocyte count above 1*10 9 /L and platelet count above 100*10 9 /L. 15

| Data collection
Baseline clinical data of AML patients were recorded after initial evaluation was completed, and the main clinical data included age, gender, French-American-British (FAB) classification, cytogenetics, molecular genetics, risk stratification, and white blood cell (WBC) count. The risk stratification evaluation was based on cytogenetics and molecular genetics, according to the NCCN Guidelines (version 1.2014). Remission status of patients was documented after response assessment. Besides, survival data were collected by conventional follow-up, which was performed through regular re-examination, clinic visit or telephone calls. All patients were consecutively followed up to December 31, 2018, resulting in a median follow-up duration of 19.0 months. Event-free survival (EFS) was defined as the time interval from initiation of treatment to disease recurrence, progression, or death. Overall survival (OS) was defined as the time interval from initiation of treatment to death. For the patients not known to have disease recurred, progressed, or died at last follow-up date, they were censored on the data of last visit or on the date of last known to be alive.

| Determinations after transfection
At 24 hours after transfection, the RT-qPCR was carried out in all  The EFS and OS were illustrated by Kaplan-Meier (K-M) curves, and the differences of EFS and OS between lncRNA MVIH high group and lncRNA MVIH low group were determined by log-rank test. P value < .05 was considered as significant.

| Comparison of lncRNA MVIH expression between AML patients and controls
LncRNA MVIH expression was increased in AML patients (me- where AUC reached the maximum, and the sensitivity as well as specificity at best cutoff value were 86.3% and 51.4%, respectively ( Figure 1B).

| Correlation of lncRNA MVIH expression with FAB classification and risk stratification in AML patients
Regarding AML patients with different FAB classification, no difference of lncRNA MVIH expression was found among M1, M2, M4, M5, and M6 patients (Figure 2A,B). As to AML patients with different risk stratification, lncRNA MVIH expression was increased along with the worse risk stratification ( Figure 2C,D).

| Correlation of lncRNA MVIH expression with CR achievement and survival profiles in AML patients
In AML patients, 166 (78.3%) patients achieved CR and 46 (21.7%) patients did not achieve CR. LncRNA MVIH expression was decreased in CR patients compared to non-CR patients ( Figure 3A,B). Furthermore, both EFS (P < .001) ( Figure 3C) and OS (P = .017) ( Figure 3D) were shorter in lncRNA MVIH high-expression patients compared to lncRNA MVIH low expression patients.

| Effect of lncRNA MVIH on AML cell activities
To explore the influence of lncRNA MVIH on cell activities in AML, we conducted some in vitro experiments. RT-qPCR assay showed that lncRNA MVIH expression was increased in KG-1 (P < .001), ME-1 (P < .05), and HT-93 cells (P < .01) compared to normal BMMCs, while no difference of lncRNA MVIH expression was found between HL-60 cells and normal BMMCs (P > .05) ( Figure 4A). Since the numerically highest lncRNA MVIH expression was observed in KG-1 cells, we transfected lncRNA MVIH KD plasmids into KG-1 cells, and lncRNA MVIH expression was dramatically decreased in KD-MVIH group compared to KD-NC group (P < .001) ( Figure 4B).

For cell proliferation, it was reduced in KD-MVIH group compared
to KD-NC group at 72 hours (P < .05) ( Figure 4C). For cell apoptosis, its rate was elevated in KD-MVIH group compared to KD-NC group (P < .01) ( Figure 4D,4). Furthermore, the numerically lowest lncRNA

| D ISCUSS I ON
LncRNAs are able to regulate gene expressions at various levels, such as chromatin modification, transcription, and post-transcriptional processing. 8 Increasing studies have revealed that lncRNAs play crucial regulatory roles in various pathological processes, especially in tumorigenesis. 13 Remarkably, lncRNA MVIH, a newly found lncRNA that is initially reported to be upregulated in HCC, has been further explored and reported to be involved in the pathology of several cancers. 9,12-14 For example, lncRNA MVIH enhances cell proliferation and invasion through regulating matrix metalloproteinase (MMP) 2 and MMP9 protein expressions in NSCLC cells. 12 Also, lncRNA MVIH facilitates cell proliferation and suppresses cell apoptosis via inhibiting miR-199a in HCC cells. 13 Besides, lncRNA MVIH activates tumor-inducing angiogenesis via suppressing the secretion of phosphoglycerate kinase 1 (PGK1) in HCC cells. 14 These data reveal that lncRNA MVIH may regulate some genes or enzymes such as miR-199a, MMP9, and PGK1 to contribute to the initiation and progression of several solid tumors.
Other than the explorations of lncRNA MVIH in the pathology of solid tumors, a few clinical practices also disclose the lncRNA MVIH expression in some tumor tissues and the correlation of lncRNA MVIH expression with disease conditions in patients with solid tumors. [10][11][12][13][14] For instance, some studies display that lncRNA MVIH expression is elevated in HCC tissues as well as breast cancer tissues than that in adjacent non-cancerous tissues. 11,14 Additionally, a study discloses that lncRNA MVIH expression is positively correlated with Karnofsky performance score and World Health Organization (WHO) grade in glioma patients. 10  The predictive role of lncRNA MVIH for prognosis in solid tumors has also been disclosed in some clinical practices. [10][11][12] For instance, lncRNA MVIH high expression is associated with poor disease-free survival and OS in breast cancer patients. 11 Also, lncRNA MVIH high  To explore how lncRNA MVIH regulate cell activities in cancers, some in vitro or in vivo experiments are conducted by previous studies. 10,11 For example, lncRNA MVIH upregulation enhances cell proliferation, migration, and invasion but inhibits cell apoptosis in glioma cells. 10 Another study displays that upregulated lncRNA MVIH promotes cell proliferation but inhibits cell apoptosis in breast cancer cells. 11 However, evidence about the regulatory role of lncRNA MVIH in AML cells is limited. In our study, we found that lncRNA MVIH expression was elevated in several AML cell lines compared to normal BMMCs. Moreover, lncRNA MVIH promoted cell proliferation but reduced cell apoptosis in AML cell lines. These results might be on account of that lncRNA MVIH regulated some genes such as jun-B and miR-199a, which have been implicated as crucial regulators in the pathology of AML, and eventually enhanced AML cell proliferation but inhibit apoptosis. 16,17 Most importantly, our data implied that lncRNA MVIH might act as a possible treatment target in AML.
In conclusion, lncRNA MVIH may not only serve as a prognostic marker but also act as a therapeutic target in AML.

ACK N OWLED G M ENTS
None.